Hepatitis B virus (HBV) covalently closed circular (ccc) DNA plays a central role in the establishment of viral infection and persistence, and is the basis for viral rebound after the cessation of therapy, as well as the elusiveness of a cure even after extended treatment with current approved medications. HBV cccDNA is established upon initial infection through conversion of the partially double stranded relaxed circular (rc) DNA virl genome, presumably through employment of the host cell's DNA repair mechanisms in the nucleus. The cccDNA episome levels are maintained through a replication pathway that involves retrotranscription of a cccDNA transcript, termed pregenomic RNA, into progeny rcDNA genomes, some of which are returned to the nucleus for conversion into cccDNA. The conversion of rcDNA into cccDNA requires the removal of a covalently-linked copy of the polymerase from the 5' end of one of the DNA strands, and this deproteinization step generates a DNA intermediate, the deproteinized rcDNA (DP-rcDNA), as precursor for cccDNA formation. In addition, our previous work suggested that the rcDNA deproteinization is a trigger signal for transportation of HBV nucleocapsid containing mature viral DNA into nucleus, where the rcDNA to cccDNA conversion takes place. However, there are many details yet to be elucidated in the understanding of cccDNA formation and metabolism. In this research application, by making use of a battery of molecular biology, biochemistry, and proteomics technologies specially designed for cccDNA study, we propose to further characterize the terminal structure and the state of existence of both cytoplasmic and nuclear DP-rcDNA, elucidate the molecular mechanisms of rcDNA deproteinization, and systematically identify host DNA repair genes involved in rcDNA to cccDNA conversion. Our ultimate goal is to illustrate a coherent picture of the molecular mechanisms/pathway for HBV cccDNA formation. The accomplishment of this project will fill a significant knowledge gap in HBV molecular biology, and potentially provide new antiviral targets for development of novel therapeutics for hepatitis B.